ABSTRACT
Coculturing of bone-forming and blood vessel-forming cells is a strategy aimed at increasing vascularity of implanted bone constructs in tissue-engineering applications. We previously described that the coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves the differentiation of both cell types, leading to the formation of functional blood vessels and enhanced bone regeneration. The objective of this study was to further delineate the multifaceted interactions between both cell types. To investigate the proteome of hOBs after cocultivation with HUVECs we used stable isotope labeling by amino acids in cell culture, revealing 49 significantly upregulated, and 54 significantly downregulated proteins. Amongst the highest regulated proteins, we found the proteins important for osteoblast differentiation, cellular adhesion, and extracellular matrix function, notably: connective tissue growth factor, desmoplakin, galectin-3, and cyclin-dependent kinase 6. The findings were confirmed by enzyme-linked immunosorbent assays. We also investigated whether the mRNA transcripts correlate with the changes in protein levels by quantitative real-time reverse transcription polymerase chain reaction. In addition, the data was compared to our previous microarray analysis of hOB transcriptome. Taken together, this in-depth analysis delivers reliable data suggesting the importance of coculturing of hOBs and HUVECs in tissue engineering.
Subject(s)
Cell Differentiation/physiology , Extracellular Matrix/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Osteoblasts/metabolism , Proteomics/methods , Blood Proteins , Bone Regeneration , Cells, Cultured , Coculture Techniques/methods , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Desmoplakins/genetics , Desmoplakins/metabolism , Down-Regulation/genetics , Galectin 3/genetics , Galectin 3/metabolism , Galectins , Humans , Osteogenesis , RNA, Messenger/genetics , Tissue Engineering/methods , Transcription, Genetic , Up-Regulation/geneticsABSTRACT
It is a common complication to develop a secondary lymphedema after surgery or radiation, for example, after axillary lymph node dissection due to breast cancer and current therapies are mainly symptomatic. Since these surgical procedures result in both, loss of adipose tissue and loss of lymphatic nodes and vessels, tissue engineering could be a new promising approach, to create an adipose tissue substitute comprised with a lymphatic network. We have conducted co-culture experiments to investigate the effects of human adipose-derived stem cells (ASCs) on human lymphatic endothelial cells (LECs) in terms of gene expression profile, proliferation, migration, and tube formation in vitro. In this respect, both cell types were co-cultured either indirectly or directly with or without the recombinant growth factor VEGF-C. Indirect co-cultures were performed with the aid of a transwell chamber. In case of direct co-culture, immunomagnetic separation by CD31 magnetic beads allowed examination of the LEC population. Direct and indirect co-culture of ASCs induced mRNA expression of lymphatic marker genes, proliferation, and migration by LECs without affecting tube formation. Thus, we have shown that co-culture of ASCs with LECs might be a feasible approach that could be used in cell-based tissue engineering therapies to heal or improve a secondary lymphedema. J. Cell. Biochem. 117: 2620-2629, 2016. © 2016 Wiley Periodicals, Inc.
